The effect of low-dose X-irradiation on numerical and structural chromosome anomaly induction in mouse immature oocytes

The ability of low doses of X-rays to induce numerical and structural chromosome anomalies in immature oocytes was examined in two experiments. In the first, 10-11-day-old females were given 0.1 or 0.2 Gy of X-rays and sampled at intervals up to 32 weeks later. In the second, 4-5-week-old females were given 0.1 Gy of X-rays and sampled up to 36 weeks post-irradiation. Chromosome anomalies were assessed in metaphase II oocytes. In the first experiment, there was evidence of dose-related increases in both hyperhaploidy (n + 1) and structural chromosome anomalies. In the second experiment, only the frequency of structural chromosome anomalies was found to increase consistently after irradiation. There was no indication that radiation-induced depletion of the oocyte population was associated with an early onset of the maternal age effect on nondisjunction.     

Elimination of X-ray-induced chromosomal aberrations in the progeny of female mice

Female NMRI mice were irradiated with various doses of X-rays and induced chromosome aberrations were scored in MII oocytes (Dosage: 0.222, 0.666, 2 and 6 Gy). After irradiation with 2 Gy, early zygotes were examined in the 2-cell stage; additional dominant lethals were counted and surviving embryos were examined after 13.5 days of pregnancy. 87.2% of the MII oocytes showed structural chromosomal aberrations after irradiation with 2 Gy. Surviving embryos, however, failed to show any increase in the aberration rate. This result points to (almost) complete elimination of genetically damaged oocytes and zygotes already before birth. In addition to the structural aberrations, aneuploidies were induced. Most of them, however, were hypoploidies. Hence, the study confirmed the well-known susceptibility of oocytes around the time of fertilization for induced chromosome loss. Induced hyperploidies, however, were very rare. Evidence for induction of meiotic non-disjunction was weak. In surviving embryos, no increase in numerical aberrations, either hypoploid or hyperploid was discovered. The significance of these data for the prediction of chromosomal damage due to to ionizing radiation in humans is discussed. Recent risk estimates of UNSCEAR and other agencies represent very cautious upper levels.     

Chromosome malsegregation and embryonic lethality induced by treatment of normally ovulated mouse oocytes with nocodazole

The mouse egg is ovulated with its nucleus arrested at the metaphase-II stage of meiosis. Sperm entry triggers the completion of the second meiotic division. It has been speculated that damage to the meiotic spindle of normally ovulated eggs at around the time of sperm entry could result in chromosome malsegregation and the death of conceptuses with numerical chromosome anomalies. This hypothesis was tested using nocodazole, a microtubule inhibitor. Nocodazole was administered either to maturing preovulatory oocytes or to normally ovulated eggs at one of the following stages: (1) the time of sperm entry, (2) early pronuclear stage, (3) pronuclear DNA synthesis, (4) prior to first cleavage division, (5) early 2-cell stage, or (6) prior to the second cleavage division. Little or no effect was observed for treatment times other than the time of sperm entry, when the egg is being activated to complete the second meiotic division. Remarkably high frequencies of embryonic lethality, expressed at around the time of implantation, were induced at this stage. Cytogenetic analysis of first cleavage metaphases of zygotes treated at the time of sperm entry revealed a high incidence of varied numerical chromosome anomalies, with changes in ploidy being predominant.     

Gamma-ray-induced numerical and structural chromosome anomalies in mouse immature oocytes

Young female mice were given 1, 2 or 3 Gy of chronic gamma-irradiation. Metaphase II oocytes from these mice were sampled 8 weeks after the end of the treatment and screened for numerical and structural chromosome anomalies. The proportions of hyperhaploid (n + 1) metaphase II oocytes increased after 1 and 2 Gy (significantly after the latter) but remained at the control level after 3 Gy of gamma-rays. Structural chromosome anomalies were significantly increased above control levels at all doses and also showed an increase with dose to 2 Gy and a decline at 3 Gy. The cause of this unusual dose-response pattern for induced chromosome damage is uncertain. These results show that significant chromosome damage can be induced by irradiation of immature oocytes, a cell stage previously suggested to be resistant to induced genetic damage.     

Absence of correlation between univalent formation and meiotic nondisjunction in aged female Chinese hamsters

The effects of maternal aging on the configuration of chiasmata, formation of univalents, and segregation of first meiotic (MI) chromosomes were investigated in young (5-8 mo) and old (16-19 mo) Chinese hamsters. Primary oocytes were collected only from mature follicles approximately 10 h before ovulation, and secondary oocytes were obtained from the oviducts 5 h after spontaneous ovulation. The average number of chiasmata per oocyte was significantly smaller in aged hamsters than in the young hamsters (P less than 0.001). Terminal chiasmata were found more frequently in the former group than in the latter one (P less than 0.001). These results coincided well with findings in the mouse. Since the 11 meiotic chromosomes could be divided into four morphologically distinguishable subgroups, it was possible to determine whether the same bivalent forming univalents at MI actually underwent nondisjunction in the following meiotic division. The incidence of both MI oocytes with a univalent pair and aneuploid MII oocytes due to first meiotic nondisjunction was significantly higher in the aged group than in the young group (P less than 0.01) and P less than 0.05, respectively). However, univalents occurred almost exclusively in the smallest metacentric chromosome group (96%), whereas nondisjunction took place nearly equally in each chromosomal subgroup. These results clearly showed that there was no correlation between the univalents seen at MI and nondisjunction during the first meiotic division.     

Chromosome anomalies in mouse oocytes after irradiation.

We investigated the cytogenetic effects of X-rays on unfertilized mouse oocytes. NMRI females received an irradiation with 0,22.2,66.6,200, and 600 R during the preovulatory phase 3 hrs after HCG (human chorionic gonadotrophin). This is a stage during oogenesis in which the oocytes pass from late dictyotene to diakinesis. Chromosome analysis was performed after ovulation at metaphase II. From these experiments we can draw the following conclusions: 1) X-rays induced during the preovulatory phase a high number of chromosome anomalies. Among these, structural anomalies prevail. 7 out of 144 ovulated oocytes in matched controls carried such an abnormality, whereas after irradiation we observed with 22.2, 66.6, 200, and 600 R, 11 out of 72, 34 out of 108, 89 out of 102, and 122 out of 124, respectively. 2) Irradiation seems also to affect the chromosome segregation during the 1. meiotic division, as we observed after 22.2, 66.6, and 200 R a total of 6 oocytes out of 204 with a supernummary chromosome. In controls, however, no hyperploidy was found in 143 ova. This increase, however, was not significant. 3) Chromosome anomalies, e.g. breaks and deletions that go back to a one-break event increased linearly with increasing dose. Exchanges, however, going back to two-break events fittest best to the linear-quadratic dose-response model. 4) The dose of 600 R seems to represents a kind of borderline in this experiment, because nearly all (122 out 124) carried at least one structural chromosome anomaly. It is also this dose after which the highest frequency of reciprocal translocations was observed in a hump-shaped slope in spermatocytes after irradiation of spermatogonia (Preston and Brewen, 1973). With an increasing dosage up to 1200 R the frequency of translocations decrease again. The elimination of cells, crossing this borderline, might be due to genetic or non-genetic effects. 5) The frequency of radiation-induced translocations per oocyte agrees with the frequency of translocations in human lymphocytes (Dolphin and Lloyd, 1974) after in vitro irradiation. 6) Significant, lower frequencies of structural chromosome anomalies were observed irradiating earlier stages of mouse oogenesis. These stages are dictyotene from females at the age of 3, or 6 weeks and prophase I-stages in female embryos on the 17th day of gestation. This result may be due to a lower sensitivity of these stages or to modifying events during the interval between irradiation and preparations.     

Spontaneous and sperm-induced activation of oocytes in LT/Sv strain mice

The oocytes of LT/Sv strain mice are unique in that a high proportion of them (∼40% in this study) are ovulated before reaching metaphase of the second meiotic division (metaphase II). The remaining oocytes of LT/Sv mice are ovulated at metaphase II, as in other strains of mice. When recently ovulated oocytes were cultured in vitro for 11–12 h, those ovulated at metaphase II remained at this stage, whereas those ovulated at metaphase of the first meiotic division (metaphase I) commonly resumed meiosis during in vitro aging. These oocytes extrude the polar body and form a diploid pronucleus. This oocyte activation is not coupled with cortical granule exocytosis. The oocytes ovulated at metaphase II are fully capable of normal fertilization, whereas those ovulated at metaphase I are not. Approximately 50% of metaphase I oocytes penetrated by spermatozoa remain at this stage, and sperm nuclei frequently undergo premature chromosome condensation. Only 13% of spermpenetrated metaphase I oocytes formed a diploid female pronucleus and a haploid male pronucleus by 4 h after insemination. These results demonstrate that the two types of ovulated LT/Sv oocytes have different potentials to undergo either spontaneous or sperm-induced activation.     

Chromosome malsegregation induced by the rodent carcinogens acetamide,pyridine and diethanolamine in Drosophila melanogaster females

The effect of the rodent carcinogens acetamide (AC), pyridine (PY) and diethanolamine (DEA) on meiotic chromosome segregation was assessed in 4-day-old Drosophila melanogaster females. After oral treatment with 0.05, 0.1, 0.2 and 0.3% PY; 0.5, 1, 1.5, 2 and 4% AC; or 5, 10, 20, 40 and 80% DEA, the females were mated to 7-day-old males and three 24h broods were obtained to sample cells exposed mainly as mature oocytes (brood I), and nearly mature oocytes (brood II) with an increasing proportion of early oocytes (brood III). Viability was not affected at the two (PY) or three (AC, DEA) lowest concentrations, decreasing thereafter. PY increased the frequency of nondisjunction exclusively in brood II suggesting its interaction with specific targets. AC and DEA (the most active of the three) induced similar frequencies of nondisjunction in all broods suggesting unspecific cell division perturbations probably due to toxicity. No clear dose effect relationships were observed.     

Analysis of aneuploidy rate in antral and ovulated mouse oocytes during female aging

Two forms of oocytes termed SN (surrounded nucleolus) and NSN (nonsurrounded nucleolus) differing for the spatial distribution of nuclear and nucleolar-associated chromatin have been described within the antral compartment of the ovary of a number of mammals. The biological significance of these two kind of oocytes is as yet not completely clear. In previous studies we have shown that prior to ovulation, mouse SN oocytes isolated from the antral compartment, matured and fertilized in vitro have a far better meiotic and developmental competence than NSN oocytes. Immediately after ovulation SN and NSN oocytes remaining in the antral compartment do not develop beyond the 2-cell stage. To further examine the correlation between chromatin distribution and meiotic competence of mouse antral oocytes, in the present study we have analyzed chromosome segregation at the first meiotic division in antral (SN and NSN) and in ovulated oocytes. SN and NSN oocytes were isolated before (48 h post PMSG injection) or after (15 h post–hCG injection) ovulation from ovaries of females of increasing age, they were cultured in vitro to metaphase II, and their aneuploidy rate was examined. Comparison of data obtained before and after ovulation highlights two main points: 1. Following ovulation a statistically significant increase of aneuploidy is observed in antral oocytes in most age groups and it is attributable to SN oocytes. 2. The aneuploidy rate of ovulated oocytes does not increase during female aging. We have found a correlation between chromatin distribution, hormonal status, and the incidence of aneuploidy during the oocyte first meiotic division. Mol. Reprod. Dev. 50 :305–312, 1998. © 1998 Wiley-Liss, Inc.     

Ultrastructural analysis of abnormalities in the morphology of the second meiotic spindle in ethanol-induced parthenogenones

A high frequency of parthenogenetic activation occurs when ovulated mouse oocytes are briefly exposed to a dilute solution of ethanol in vitro. Cytogenetic analyses of parthenogenones at metaphase of the first cleavage division have confirmed that parthenogenetic activation, per se, does not increase the incidence of chromosome segregation errors during the completion of the second meiotic division. Ethanol-induced activation, however, significantly increases the incidence of aneuploidy. The ultrastructural changes that occur in the morphology and organization of the second meiotic spindle apparatus in ethanol- and hyaluronidase-activated oocytes is reported here. Abnormalities in the arrangement of microtubule arrays and chromosome position were principally observed in ethanol-activated oocytes at anaphase and telophase of the second meiotic division, but were only rarely observed in hyaluronidase-activated oocytes. It is proposed that the abnormalities in spindle morphology and chromosome displacement observed in ethanol-activated oocytes represent the initial events that lead to chromosome segregation errors following exposure to this agent.     

Study of external low irradiation dose effects on induction of chromosome aberrations in Pisum sativum root tip meristem

The effects of low doses of ionizing radiation have been a matter of important debate over the last few years. The point of discussion concerns the validity of the linear dose-response extrapolation for low doses, used by international organizations, to establish radio-protection norms. Here, we contributed to this discussion by investigating the induction of chromosome aberrations by low to moderate doses ranging from 0 to 10 Gy in root meristem cells of 6-day-old Pisum plantlets. After acute irradiation of plantlets by a (60)Co source, the percentage of root tip meristem cells displaying chromosome aberrations was estimated immediately after irradiation and after 20 h recovery time. The dose-effect curves show non-linear responses, especially in the low dose range (0- 1 Gy), which is of particular interest. After 20 h of recovery, a steep increase of aberrations was observed for cells exposed to 0.4 Gy, followed by a plateau for doses until 1 Gy. There was an irradiation effect on plant growth during the first and second generations, showing the persistence of cell division anomalies as a long term effect of acute irradiation. This result suggests the induction of a genomic instability.Our results, in agreement with some obtained in animals, show rather non-linear dose-effect responses, with notably higher biological effects of low doses than expected.     

Ovulation and fertilization of primary and secondary oocytes in LT/Sv strain mice

In this study, the chromosome constitution of both unfertilized oocytes and fertilized eggs isolated from the oviducts of LT/Sv strain mice were analyzed. Air-dried chromosome preparations from unfertilized oocytes revealed that about one-third of those examined were ovulated as primary oocytes. These were arrested at metaphase of the first meiotic division and exhibited the characteristic “tetrad” chromosome configuration. The remaining two-thirds of the unfertilized oocytes were ovulated at metaphase of the second meiotic division. The fertilized eggs were isolated from the oviducts of LT/Sv females previously mated to (C57BL × CBA) F1 hybrid males. Analysis of the fertilized eggs at metaphase of their first cleavage mitosis revealed that about one-third of the eggs examined were digynic triploids, whereas the remaining two-thirds had the normal diploid chromsome constitution. In the triploids, the 40 female chromosomes present (mouse, n = 20) were derived from a single diploid pronucleus formed after the extrusion of a first polar body, and following the monospermic fertilization of primary oocytes. The female pronuclear-derived chromosomes invariably exhibited “homologous pairing,” and these were associated at their centromeres. The ovulation, penetration, and subsequent fertilization of primary oocytes is an extremely unusual phenomenon in mammals and only appears to occur on a regular basis in LT/Sv mice. The premature “cytoplasmic maturation” of these oocytes is of interest, as they clearly have the same developmental capacity as secondary oocytes. The significance of these observations in relation to folliculogenesis and litter size in LT/Sv mice is discussed.     

Origin of the extra chromosome in trisomy 21

Summary Of 61 families of children with trisomy 21, polymorphism of chromosome 21 elucidating the origin of the extra chromosome was found in 42. Nondisjunction was of paternal origin in 8 cases (19.04%) and the anomaly occurred with equal frequency during the first and second meiotic divisions. Maternal nondisjunction was demonstrated in 34 cases (80.95%), in which nondisjunction occurred by far the most often during the first meiotic division (29 cases).These results are in agreement with data from the literature, and suggest the existence of at least two different causes for chromosomal nondisjunction, the first being the same in both sexes and occurring in both meiotic divisions and the second specifically limited to the first meiotic division in the mother.Attachée de Recherche au CNRSAttachée de Recherche à l'INSERM     

Reduced recombination and paternal age effect in Klinefelter syndrome

Summary The parental origin of the additional sex chromosome was studied in 47 cases with an XXY sex chromosome consitution. In 23 cases (49%), the error occurred during the first paternal meiotic division. Maternal origin of the additional chromosome was found in the remaining 24 cases (51%). Centromeric homo- versus heterozygosity could be determined in 18 out of the 24 maternally derived cases. According to the centromeric status and recombination rate, the nondisjunction was attributable in 9 cases (50%) to an error at the first maternal meiotic division, in 7 cases (39%) to an error at the second maternal meiotic division and in 2 cases (11%) to a nullo-chiasmata nondisjunction at meiosis II or to postzygotic mitotic error. No recombination, and in particular none in the pericentromeric region, was found in any of the 9 cases due to nondisjunction at the first maternal meiotic division. Significantly increased paternal age was found in the paternally derived cases. Maternal age was significantly higher in the maternally derived cases due to a meiotic I error compared with those due to a meiotic II error. There were no significant clinical differences between patients with respect to the origin of the additional X chromosome.     

Flow cytometric analysis of the effects of 0.4 MeV fission neutrons on mouse spermatogenesis

(C57Bl/Cne X C3H/Cne)F1 male mice were irradiated with single acute doses of 0.4 MeV neutrons ranging from 0.05 to 2 Gy, and testis cell suspensions were prepared for cytometric analysis of the DNA content 2-70 days after irradiation. Various cell subpopulations could be identified in the control histogram including mature and immature spermatids, diploid spermatogonia and spermatocytes, tetraploid cells and cells in the S-phase. Variations in the relative proportions of different cell types were detected at each dose and time, reflecting lethal damage induced on specific spermatogenetic stages. The reduction of the number of elongated spermatids 28 days after irradiation was shown to be a particularly sensitive parameter for the cytometrical assessment of the radiosensitivity of differentiating gonia. A D0 value of 0.13 Gy was calculated and compared with data obtained after X-irradiation, using the same experimental protocol. In the latter case a biphasic curve was obtained over the dose range from 0.25 to 10 Gy, possibly reflecting the existence of some cell population heterogeneity. RBE values were estimated at different neutron doses relative to the radiosensitive component of the X-ray curve, and ranged from 3.3 to 4, in agreement with data in the literature. Genotoxic effects were monitored 7 days after irradiation by a dose-dependent increase of the coefficient of variation (CV) values of the round spermatid peak, reflecting the induction of numerical and structural chromosome aberrations, and 14 or 21 days after irradiation by the detection of diploid elongated spermatids, probably arising from a radiation-induced complete failure of the first or second meiotic division.     

Incidence of nondisjunction in mouse oocytes.

The chromosomes of more than 3000 ovulated mouse oocytes from strains C3H/Han, NMRI/Han, and (101 X C3H)F1 have been analyzed after spontaneous and hormonally induced ovulation. No significant difference in the incidence of nondisjunction was observed among the three strains with or without hormonal pretreatment. The incidence of nondisjunction was estimated to be 0.47% in NMRI/Han, 0.62% in C3H/Han, and 0.81% in (101 X C3H/F1. The incidence of chromosomal imbalance after the first meiotic division was slightly higher after adding the events following presegregation. Diploidy-spontaneous and hormonally induced-reached a significant leve in NMRI/Han. This may be interpreted as a consequence of hormonal interference with a genetically defined malfunction of gene product(s) during the late phase of oogenesis.     

Meiotic arrest and aneuploidy induced by vinblastine in mouse oocytes

Young superovulated female mice were injected i.p. with single doses of vinblastine sulfate just before the onset of the first meiotic division. Secondary oocytes, fixed one by one on a slide, were cytogenetically scored. Evidence of the meiotic arresting activity of vinblastine was produced by the observation of increasing frequencies of M1-arrested oocytes and by the presence of undegenerated chromosome sets of first polar bodies. When the first meiotic division could be undertaken chromosome malsegregation occurred with high frequency, both in terms of aneuploidy and polyploidy. M1-blocked and polyploid oocytes have been interpreted as the consequence of irreversible damage to the spindle induced by vinblastine through its binding on tubulin low-affinity sites; this reaction, in fact, causes microtubule crystallization. According to this mechanism, dose-effect relationships of both phenomena show a threshold at 0.45 mg/kg. On the other hand, the incidence of aneuploid oocytes is correlated with meiotic delay, as detected by the delayed degeneration of polar bodies, and increases linearly with dose. Both phenomena are, therefore, stochastic and can be referred to the binding of the chemical on tubulin high-affinity sites, which is known to cause tubulin depolymerization in a colchicine-like way.     

高能电子束超剂量照射与效应关系的探讨——人周血淋巴细胞核损伤指标的比较观察

为了探讨在大剂量辐射条件下建立生物量计的可能,本实验研究了在10MeV电子束照射后,人体外周血淋巴细胞各种核损伤指标的改变与剂量(0—40Gy)间的关系,结果表明,随着照射剂量的增大,复合核损伤指标——核异常率、微核率、核固缩率和核裂解率亦随之上升,在0—20Gy范围内,与剂量呈线性关系,相关系数显著性检验P<0.01的核损伤指标是:核异常率、微核率。P<0.05的是核变形率;在0—40Gy范围内,与之呈线性关系,P<0.01的仅有核固缩率。和染色体畸变分析相比,核异常检测方法简便,可反映超剂鞋(如>10Gy)辐射的损伤,值得引起研究者的注意。     

The mechanism of secondary nondisjunction in Drosophila melanogaster females

Bridges (1916) observed that X chromosome nondisjunction was much more frequent in XXY females than it was in genetically normal XX females. In addition, virtually all cases of X nondisjunction in XXY females were due to XX <--> Y segregational events in oocytes in which the two X chromosomes had failed to undergo crossing over. He referred to these XX <--> Y segregation events as "secondary nondisjunction." Cooper (1948) proposed that secondary nondisjunction results from the formation of an X-Y-X trivalent, such that the Y chromosome directs the segregation of two achiasmate X chromosomes to opposite poles on the first meiotic spindle. Using in situ hybridization to X and YL chromosomal satellite sequences, we demonstrate that XX <--> Y segregations are indeed presaged by physical associations of the X and Y chromosomal heterochromatin. The physical colocalization of the three sex chromosomes is observed in virtually all oocytes in early prophase and maintained at high frequency until midprophase in all genotypes examined. Although these XXY associations are usually dissolved by late prophase in oocytes that undergo X chromosomal crossing over, they are maintained throughout prophase in oocytes with nonexchange X chromosomes. The persistence of such XXY associations in the absence of exchange presumably facilitates the segregation of the two X chromosomes and the Y chromosome to opposite poles on the developing meiotic spindle. Moreover, the observation that XXY pairings are dissolved at the end of pachytene in oocytes that do undergo X chromosomal crossing over demonstrates that exchanges can alter heterochromatic (and thus presumably centromeric) associations during meiotic prophase.     

The meiotic stage of preovulatory oocytes in mares

Confusion exists as to whether the oocytes of the domestic horse are ovulated at the first meiotic metaphase (MI) or the second (MII). In this study eight oocytes were collected from the preovulatory follicles of 16 mares 36 h after human chorionic gonadotropin CG treatment. Six of the eight oocytes were judged to be at MII by the presence of the first polar body and this judgement was confirmed by semithin sectioning in one. Of the two that had no polar body, one was found to be at MII after fixation for chromosomal analysis and the meiotic stage of the other remained undetermined. Since all seven oocytes yielding conclusive evidence were at MII, it was concluded that horse oocytes, like those of most mammals studied, are ovulated after completion of the first meiotic division and formation of the first polar body.